The fork protection complex promotes parental histone recycling and epigenetic memory.

The inheritance of parental histones across the replication fork is thought to mediate epigenetic memory. Here, we reveal that fission yeast Mrc1 (CLASPIN in humans) binds H3-H4 tetramers and operates as a central coordinator of symmetric parental histone inheritance. Mrc1 mutants in a key connector domain disrupted segregation of parental histones to the lagging strand comparable to Mcm2 histone-binding mutants. Both mutants showed clonal and asymmetric loss of H3K9me-mediated gene silencing. AlphaFold predicted co-chaperoning of H3-H4 tetramers by Mrc1 and Mcm2, with the Mrc1 connector domain bridging histone and Mcm2 binding. Biochemical and functional analysis validated this model and revealed a duality in Mrc1 function: disabling histone binding in the connector domain disrupted lagging-strand recycling while another histone-binding mutation impaired leading strand recycling. We propose that Mrc1 toggles histones between the lagging and leading strand recycling pathways, in part by intra-replisome co-chaperoning, to ensure epigenetic transmission to both daughter cells.

Fission yeast Swi2 designates cell-type specific donor and stimulates Rad51-driven strand exchange for mating-type switching.

A haploid of the fission yeast Schizosaccharomyces pombe expresses either the P or M mating-type, determined by the active, euchromatic, mat1 cassette. Mating-type is switched by Rad51-driven gene conversion of mat1 using a heterochromatic donor cassette, mat2-P or mat3-M. The Swi2-Swi5 complex, a mating-type switching factor, is central to this process by designating a preferred donor in a cell-type-specific manner. Swi2-Swi5 selectively enables one of two cis-acting recombination enhancers, SRE2 adjacent to mat2-P or SRE3 adjacent to mat3-M. Here, we identified two functionally important motifs in Swi2, a Swi6 (HP1 homolog)-binding site and two DNA-binding AT-hooks. Genetic analysis demonstrated that the AT-hooks were required for Swi2 localization at SRE3 to select the mat3-M donor in P cells, while the Swi6-binding site was required for Swi2 localization at SRE2 to select mat2-P in M cells. In addition, the Swi2-Swi5 complex promoted Rad51-driven strand exchange in vitro. Taken together, our results show how the Swi2-Swi5 complex would localize to recombination enhancers through a cell-type specific binding mechanism and stimulate Rad51-driven gene conversion at the localization site.

Establishment of heterochromatin in domain-size-dependent bursts.

Methylation of histone H3K9 is a hallmark of epigenetic silencing in eukaryotes. Nucleosome modifications often rely on positive feedback where enzymes are recruited by modified nucleosomes. A combination of local and global feedbacks has been proposed to account for some dynamic properties of heterochromatin, but the range at which the global feedbacks operate and the exact mode of heterochromatin propagation are not known. We investigated these questions in fission yeast. Guided by mathematical modeling, we incrementally increased the size of the mating-type region and profiled heterochromatin establishment over time. We observed exponential decays in the proportion of cells with active reporters, with rates that decreased with domain size. Establishment periods varied from a few generations in wild type to >200 generations in the longest region examined, and highly correlated silencing of two reporters located outside the nucleation center was observed. On a chromatin level, this indicates that individual regions are silenced in sudden bursts. Mathematical modeling accounts for these bursts if heterochromatic nucleosomes facilitate a deacetylation or methylation reaction at long range, in a distance-independent manner. A likely effector of three-dimensional interactions is the evolutionarily conserved Swi6HP1 H3K9me reader, indicating the bursting behavior might be a general mode of heterochromatin propagation.

Integrity of a heterochromatic domain ensured by its boundary elements.

In fission yeast, the inverted repeats IR-L and IR-R function as boundary elements at the edges of a 20-kb silent heterochromatic domain where nucleosomes are methylated at histone H3K9. Each repeat contains a series of B-box motifs physically associated with the architectural TFIIIC complex and with other factors including the replication regulator Sap1 and the Rix1 complex (RIXC). We demonstrate here the activity of these repeats in heterochromatin formation and maintenance. Deletion of the entire IR-R repeat or, to a lesser degree, deletion of just the B boxes impaired the de novo establishment of the heterochromatic domain. Nucleation proceeded normally at the RNA interference (RNAi)-dependent element cenH but subsequent propagation to the rest of the region occurred at reduced rates in the mutants. Once established, heterochromatin was unstable in the mutants. These defects resulted in bistable populations of cells occupying alternate "on" and "off" epigenetic states. Deleting IR-L in combination with IR-R synergistically tipped the balance toward the derepressed state, revealing a concerted action of the two boundaries at a distance. The nuclear rim protein Amo1 has been proposed to tether the mating-type region and its boundaries to the nuclear envelope, where Amo1 mutants displayed milder phenotypes than boundary mutants. Thus, the boundaries might facilitate heterochromatin propagation and maintenance in ways other than just through Amo1, perhaps by constraining a looped domain through pairing.

New insights into donor directionality of mating-type switching in Schizosaccharomyces pombe.

Mating-type switching in Schizosaccharomyces pombe entails programmed gene conversion events regulated by DNA replication, heterochromatin, and the HP1-like chromodomain protein Swi6. The whole mechanism remains to be fully understood. Using a gene deletion library, we screened ~ 3400 mutants for defects in the donor selection step where a heterochromatic locus, mat2-P or mat3-M, is chosen to convert the expressed mat1 locus. By measuring the biases in mat1 content that result from faulty directionality, we identified in total 20 factors required for donor selection. Unexpectedly, these included the histone H3 lysine 4 (H3K4) methyltransferase complex subunits Set1, Swd1, Swd2, Swd3, Spf1 and Ash2, the BRE1-like ubiquitin ligase Brl2 and the Elongator complex subunit Elp6. The mutant defects were investigated in strains with reversed donor loci (mat2-M mat3-P) or when the SRE2 and SRE3 recombination enhancers, adjacent to the donors, were deleted or transposed. Mutants in Set1C, Brl2 or Elp6 altered balanced donor usage away from mat2 and the SRE2 enhancer, towards mat3 and the SRE3 enhancer. The defects in these mutants were qualitatively similar to heterochromatin mutants lacking Swi6, the NAD+-dependent histone deacetylase Sir2, or the Clr4, Raf1 or Rik1 subunits of the histone H3 lysine 9 (H3K9) methyltransferase complex, albeit not as extreme. Other mutants showed clonal biases in switching. This was the case for mutants in the NAD+-independent deacetylase complex subunits Clr1, Clr2 and Clr3, the casein kinase CK2 subunit Ckb1, the ubiquitin ligase component Pof3, and the CENP-B homologue Cbp1, as well as for double mutants lacking Swi6 and Brl2, Pof3, or Cbp1. Thus, we propose that Set1C cooperates with Swi6 and heterochromatin to direct donor choice to mat2-P in M cells, perhaps by inhibiting the SRE3 recombination enhancer, and that in the absence of Swi6 other factors are still capable of imposing biases to donor choice.

Establishment of expression-state boundaries by Rif1 and Taz1 in fission yeast.

The Shelterin component Rif1 has emerged as a global regulator of the replication-timing program in all eukaryotes examined to date, possibly by modulating the 3D-organization of the genome. In fission yeast a second Shelterin component, Taz1, might share similar functions. Here, we identified unexpected properties for Rif1 and Taz1 by conducting high-throughput genetic screens designed to identify cis- and trans-acting factors capable of creating heterochromatin-euchromatin boundaries in fission yeast. The preponderance of cis-acting elements identified in the screens originated from genomic loci bound by Taz1 and associated with origins of replication whose firing is repressed by Taz1 and Rif1. Boundary formation and gene silencing by these elements required Taz1 and Rif1 and coincided with altered replication timing in the region. Thus, small chromosomal elements sensitive to Taz1 and Rif1 (STAR) could simultaneously regulate gene expression and DNA replication over a large domain, at the edge of which they established a heterochromatin-euchromatin boundary. Taz1, Rif1, and Rif1-associated protein phosphatases Sds21 and Dis2 were each sufficient to establish a boundary when tethered to DNA. Moreover, efficient boundary formation required the amino-terminal domain of the Mcm4 replicative helicase onto which the antagonistic activities of the replication-promoting Dbf4-dependent kinase and Rif1-recruited phosphatases are believed to converge to control replication origin firing. Altogether these observations provide an insight into a coordinated control of DNA replication and organization of the genome into expression domains.

Mating-type switching by homology-directed recombinational repair: a matter of choice.

In eukaryotes, all DNA transactions happen in the context of chromatin that often takes part in regulatory mechanisms. In particular, chromatin structure can regulate exchanges of DNA occurring through homologous recombination. Few systems have provided as detailed a view on this phenomenon as mating-type switching in yeast. Mating-type switching entails the choice of a template for the gene conversions of the expressed mating-type locus. In the fission yeast Schizosaccharomyces pombe, correct template choice requires two competing small recombination enhancers, SRE2 and SRE3, that function in the context of heterochromatin. These two enhancers act with the Swi2/Swi5 recombination accessory complex to initiate strand exchange in a cell-type-specific manner, from SRE2 in M cells and SRE3 in P cells. New research indicates that the Set1C complex, responsible for H3K4 methylation, and the Brl2 ubiquitin ligase, that catalyzes H2BK119 ubiquitylation, participate in the cell-type-specific selection of SRE2 or SRE3. Here, we review these findings, compare donor preference in S. pombe to the distantly related budding yeast Saccharomyces cerevisiae, and contrast the positive effects of heterochromatin on the donor selection process with other situations, where heterochromatin represses recombination.

Two portable recombination enhancers direct donor choice in fission yeast heterochromatin.

Mating-type switching in fission yeast results from gene conversions of the active mat1 locus by heterochromatic donors. mat1 is preferentially converted by mat2-P in M cells and by mat3-M in P cells. Here, we report that donor choice is governed by two portable recombination enhancers capable of promoting use of their adjacent cassette even when they are transposed to an ectopic location within the mat2-mat3 heterochromatic domain. Cells whose silent cassettes are swapped to mat2-M mat3-P switch mating-type poorly due to a defect in directionality but cells whose recombination enhancers were transposed together with the cassette contents switched like wild type. Trans-acting mutations that impair directionality affected the wild-type and swapped cassettes in identical ways when the recombination enhancers were transposed together with their cognate cassette, showing essential regulatory steps occur through the recombination enhancers. Our observations lead to a model where heterochromatin biases competitions between the two recombination enhancers to achieve directionality.

Subnuclear relocalization and silencing of a chromosomal region by an ectopic ribosomal DNA repeat.

Our research addresses the relationship between subnuclear localization and gene expression in fission yeast. We observed the relocalization of a heterochromatic region, the mating-type region, from its natural location at the spindle-pole body to the immediate vicinity of the nucleolus. Relocalization occurred in response to a DNA rearrangement replacing a boundary element (IR-R) with a ribosomal DNA repeat (rDNA-R). Gene expression was strongly silenced in the relocalized mating-type region through mechanisms that differ from those operating in wild type. Also different from the wild-type situation, programmed recombination events failed to take place in the rDNA-R mutant. Increased silencing and perinucleolar localization depended on Reb1, a DNA-binding protein with cognate sites in the rDNA. Reb1 was recently shown to mediate long-range interchromosomal interactions in the nucleus through dimerization, providing a mechanism for the observed relocalization. Replacing the full rDNA repeat with Reb1-binding sites, and using mutants lacking the histone H3K9 methyltransferase Clr4, indicated that the relocalized region was silenced redundantly by heterochromatin and another mechanism, plausibly antisense transcription, achieving a high degree of repression in the rDNA-R strain.

H3K9me-independent gene silencing in fission yeast heterochromatin by Clr5 and histone deacetylases.

Nucleosomes in heterochromatic regions bear histone modifications that distinguish them from euchromatic nucleosomes. Among those, histone H3 lysine 9 methylation (H3K9me) and hypoacetylation have been evolutionarily conserved and are found in both multicellular eukaryotes and single-cell model organisms such as fission yeast. In spite of numerous studies, the relative contributions of the various heterochromatic histone marks to the properties of heterochromatin remain largely undefined. Here, we report that silencing of the fission yeast mating-type cassettes, which are located in a well-characterized heterochromatic region, is hardly affected in cells lacking the H3K9 methyltransferase Clr4. We document the existence of a pathway parallel to H3K9me ensuring gene repression in the absence of Clr4 and identify a silencing factor central to this pathway, Clr5. We find that Clr5 controls gene expression at multiple chromosomal locations in addition to affecting the mating-type region. The histone deacetylase Clr6 acts in the same pathway as Clr5, at least for its effects in the mating-type region, and on a subset of other targets, notably a region recently found to be prone to neo-centromere formation. The genomic targets of Clr5 also include Ste11, a master regulator of sexual differentiation. Hence Clr5, like the multi-functional Atf1 transcription factor which also modulates chromatin structure in the mating-type region, controls sexual differentiation and genome integrity at several levels. Globally, our results point to histone deacetylases as prominent repressors of gene expression in fission yeast heterochromatin. These deacetylases can act in concert with, or independently of, the widely studied H3K9me mark to influence gene silencing at heterochromatic loci.

RNAi and heterochromatin repress centromeric meiotic recombination.

During meiosis, the formation of viable haploid gametes from diploid precursors requires that each homologous chromosome pair be properly segregated to produce an exact haploid set of chromosomes. Genetic recombination, which provides a physical connection between homologous chromosomes, is essential in most species for proper homologue segregation. Nevertheless, recombination is repressed specifically in and around the centromeres of chromosomes, apparently because rare centromeric (or pericentromeric) recombination events, when they do occur, can disrupt proper segregation and lead to genetic disabilities, including birth defects. The basis by which centromeric meiotic recombination is repressed has been largely unknown. We report here that, in fission yeast, RNAi functions and Clr4-Rik1 (histone H3 lysine 9 methyltransferase) are required for repression of centromeric recombination. Surprisingly, one mutant derepressed for recombination in the heterochromatic mating-type region during meiosis and several mutants derepressed for centromeric gene expression during mitotic growth are not derepressed for centromeric recombination during meiosis. These results reveal a complex relation between types of repression by heterochromatin. Our results also reveal a previously undemonstrated role for RNAi and heterochromatin in the repression of meiotic centromeric recombination and, potentially, in the prevention of birth defects by maintenance of proper chromosome segregation during meiosis.

The transcription factor Atf1 lowers the transition barrier for nucleosome-mediated establishment of heterochromatin.

Transcription factors can exert opposite effects depending on the chromosomal context. The fission yeast transcription factor Atf1 both activates numerous genes in response to stresses and mediates heterochromatic gene silencing in the mating-type region. Investigating this context dependency, we report here that the establishment of silent heterochromatin in the mating-type region occurs at a reduced rate in the absence of Atf1 binding. Quantitative modeling accounts for the observed establishment profiles by a combinatorial recruitment of histone-modifying enzymes: locally by Atf1 at two binding sites and over the whole region by dynamically appearing heterochromatic nucleosomes, a source of which is the RNAi-dependent cenH element. In the absence of Atf1 binding, the synergy is lost, resulting in a slow rate of heterochromatin formation. The system shows how DNA-binding proteins can influence local nucleosome states and thereby potentiate long-range positive feedback on histone-modification reactions to enable heterochromatin formation over large regions in a context-dependent manner.

Euchromatin factors HULC and Set1C affect heterochromatin organization and mating-type switching in fission yeast Schizosaccharomyces pombe.

Mating-type (P or M) of fission yeast Schizosaccharomyces pombe is determined by the transcriptionally active mat1 cassette and is switched by gene conversion using a donor, either mat2 or mat3, located in an adjacent heterochromatin region (mating-type switching; MTS). In the switching process, heterochromatic donors of genetic information are selected based on the P or M cell type and on the action of two recombination enhancers, SRE2 promoting the use of mat2-P and SRE3 promoting the use of mat3-M, leading to replacement of the content of the expressed mat1 cassette. Recently, we found that the histone H3K4 methyltransferase complex Set1C participates in donor selection, raising the question of how a complex best known for its effects in euchromatin controls recombination in heterochromatin. Here, we report that the histone H2BK119 ubiquitin ligase complex HULC functions with Set1C in MTS, as mutants in the shf1, brl1, brl2 and rad6 genes showed defects similar to Set1C mutants and belonged to the same epistasis group as set1Δ. Moreover, using H3K4R and H2BK119R histone mutants and a Set1-Y897A catalytic mutant, we found that ubiquitylation of histone H2BK119 by HULC and methylation of histone H3K4 by Set1C are functionally coupled in MTS. Cell-type biases in MTS in these mutants suggested that HULC and Set1C inhibit the use of the SRE3 recombination enhancer in M cells, thus favoring SRE2 and mat2-P. Consistent with this, imbalanced switching in the mutants was traced to compromised association of the directionality factor Swi6 with the recombination enhancers in M cells. Based on their known effects at other chromosomal locations, we speculate that HULC and Set1C control nucleosome mobility and strand invasion near the SRE elements. In addition, we uncovered distinct effects of HULC and Set1C on histone H3K9 methylation and gene silencing, consistent with additional functions in the heterochromatic domain.

Histone modifications: cycling with chromosomal replication.

Histone modifications tend to be lost during chromosome duplication. Several recent studies suggest that the RNA interference pathway becomes active during the weakened transcriptional repression occurring at centromeres in S phase, resulting in the re-establishment of histone modifications that direct the formation of heterochromatin.

Global effects on gene expression in fission yeast by silencing and RNA interference machineries.

Histone modifications influence gene expression in complex ways. The RNA interference (RNAi) machinery can repress transcription by recruiting histone-modifying enzymes to chromatin, although it is not clear whether this is a general mechanism for gene silencing or whether it requires repeated sequences such as long terminal repeats (LTRs). We analyzed the global effects of the Clr3 and Clr6 histone deacetylases, the Clr4 methyltransferase, the zinc finger protein Clr1, and the RNAi proteins Dicer, RdRP, and Argonaute on the transcriptome of Schizosaccharomyces pombe (fission yeast). The clr mutants derepressed similar subsets of genes, many of which also became transcriptionally activated in cells that were exposed to environmental stresses such as nitrogen starvation. Many genes that were repressed by the Clr proteins clustered in extended regions close to the telomeres. Surprisingly few genes were repressed by both the silencing and RNAi machineries, with transcripts from centromeric repeats and Tf2 retrotransposons being notable exceptions. We found no correlation between repression by RNAi and proximity to LTRs, and the wtf family of repeated sequences seems to be repressed by histone deacetylation independent of RNAi. Our data indicate that the RNAi and Clr proteins show only a limited functional overlap and that the Clr proteins play more global roles in gene silencing.

Evolutionary-conserved telomere-linked helicase genes of fission yeast are repressed by silencing factors, RNAi components and the telomere-binding protein Taz1.

In Schizosaccharomyces pombe the RNAi machinery and proteins mediating heterochromatin formation regulate the transcription of non-coding centromeric repeats. These repeats share a high sequence similarity with telomere-linked helicase (tlh) genes, implying an ancestral relationship between the two types of elements and suggesting that transcription of the tlh genes might be regulated by the same factors as centromeric repeats. Indeed, we found that mutants lacking the histone methyltransferase Clr4, the Pcu4 cullin, Clr7 or Clr8, accumulate high levels of tlh forward and reverse transcripts. Mutations and conditions perturbing histone acetylation had similar effects further demonstrating that the tlh genes are normally repressed by heterochromatin. In contrast, mutations in the RNAi factors Dcr1, Ago1 or Rdp1 led only to a modest derepression of the tlh genes indicating an alternate pathway recruits heterochromatin components to telomeres. The telomere-binding protein Taz1 might be part of such a redundant pathway, tlh transcripts being present at low levels in Deltataz1 mutants and at higher levels in Deltataz1 Deltadcr1 double mutants. Surprisingly, the chromodomain protein Chp1, a component of the Ago1-containing RITS complex, contributes more to tlh repression than Ago1, indicating the repressive effects of Chp1 are partially independent of RITS. The tlh genes are found in the subtelomeric regions of several other fungi raising the intriguing possibility of conserved regulation and function.

Structure of the replication regulator Sap1 reveals functionally important interfaces.

The mechanism by which specific protein-DNA complexes induce programmed replication fork stalling in the eukaryotic genome remains poorly understood. In order to shed light on this process we carried out structural investigations on the essential fission yeast protein Sap1. Sap1 was identified as a protein involved in mating-type switching in Schizosaccharomyces pombe, and has been shown to be involved in programmed replication fork stalling. Interestingly, Sap1 assumes two different DNA binding modes. At the mating-type locus dimers of Sap1 bind the SAS1 sequence in a head-to-head arrangement, while they bind to replication fork blocking sites at rDNA and Tf2 transposons in a head-to-tail mode. In this study, we have solved the crystal structure of the Sap1 DNA binding domain and we observe that Sap1 molecules interact in the crystal using a head-to-tail arrangement that is compatible with DNA binding. We find that Sap1 mutations which alleviate replication-fork blockage at Tf2 transposons in CENP-B mutants map to the head-to-tail interface. Furthermore, several other mutations introduced in this interface are found to be lethal. Our data suggests that essential functions of Sap1 depend on its head-to-tail oligomerization.

Dependency of Heterochromatin Domains on Replication Factors.

Chromatin structure regulates both genome expression and dynamics in eukaryotes, where large heterochromatic regions are epigenetically silenced through the methylation of histone H3K9, histone deacetylation, and the assembly of repressive complexes. Previous genetic screens with the fission yeast Schizosaccharomyces pombe have led to the identification of key enzymatic activities and structural constituents of heterochromatin. We report here on additional factors discovered by screening a library of deletion mutants for silencing defects at the edge of a heterochromatic domain bound by its natural boundary-the IR-R+ element-or by ectopic boundaries. We found that several components of the DNA replication progression complex (RPC), including Mrc1/Claspin, Mcl1/Ctf4, Swi1/Timeless, Swi3/Tipin, and the FACT subunit Pob3, are essential for robust heterochromatic silencing, as are the ubiquitin ligase components Pof3 and Def1, which have been implicated in the removal of stalled DNA and RNA polymerases from chromatin. Moreover, the search identified the cohesin release factor Wpl1 and the forkhead protein Fkh2, both likely to function through genome organization, the Ssz1 chaperone, the Fkbp39 proline cis-trans isomerase, which acts on histone H3P30 and P38 in Saccharomyces cerevisiae, and the chromatin remodeler Fft3. In addition to their effects in the mating-type region, to varying extents, these factors take part in heterochromatic silencing in pericentromeric regions and telomeres, revealing for many a general effect in heterochromatin. This list of factors provides precious new clues with which to study the spatiotemporal organization and dynamics of heterochromatic regions in connection with DNA replication.

Positioning and elongation of the fission yeast spindle by microtubule-based pushing.

In eukaryotic cells, proper position of the mitotic spindle is necessary for successful cell division and development. We explored the nature of forces governing the positioning and elongation of the mitotic spindle in Schizosaccharomyces pombe. We hypothesized that astral microtubules exert mechanical force on the S. pombe spindle and thus help align the spindle with the major axis of the cell. Microtubules were tagged with green fluorescent protein (GFP) and visualized by two-photon microscopy. Forces were inferred both from time-lapse imaging of mitotic cells and, more directly, from mechanical perturbations induced by laser dissection of the spindle and astral microtubules. We found that astral microtubules push on the spindle poles in S. pombe, in contrast to the pulling forces observed in a number of other cell types. Further, laser dissection of the spindle midzone induced spindle collapse inward. This offers direct evidence in support of the hypothesis that spindle elongation is driven by the sliding apart of antiparallel microtubules in the spindle midzone. Broken spindles recovered and mitosis completed as usual. We propose a model of spindle centering and elongation by microtubule-based pushing forces.